Vol. 166, No. 3, 1990 February 14, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1228-1236
BIOCHEMICAL
PREPARATION AND CHARACTERIZATION OF POLYCLONAL AND MONOCLONAL ANTIBODIES AGAINST THE INSECTICIDE DDT Daniel
Biirgisser,
Stefan Frey, Stephan Klauser
Bernd
Gutte
Biochemisches
Institut der Universitat Winterthurerstrasse 190, CH-8057 Ziirich, Switzerland
Received
December
and
Ziirich,
4, 1989
A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared antiDDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/l were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site. O1990 Academic Press,Inc. Although diction
is
the still
been reported ratory
design
of
in
infancy,
(l-6).
was the
The proposed
design
B-sheet
by CD measurements, various
peptide
action
between
unpublished designed
24-residue
a naturally
of
structure and the
of
are
a monoclonal
occurring
attempts
have
this
from
labo-
field
peptide
our
polypeptide (2,7)
(2).
was confirmed
of DDT binding
studies
using
the
mode of
inter-
postulated
protein of
pre-
successful
DDT-binding
this
supported
NMR studies of
to
results
polypeptide
based on structure
several
a 24-residue
DDT and binding
results).
proteins
One contribution
analogues
The availability ting
its
novel
(2,7)(S. the
Klauser
solution
et
al.,
structure
of
in progress. anti-DDT
DDT-binding
protein,
antibody, would
represengreatly
Abbreviations: DDE,
DDT, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane; l,l-dichloro-2,2-bis(p-chlorophenyl)ethylene; DDA,
bis(p-chlorophenyl)acetic acid: Kelthan, l,l,l-trichloro-2-hydroxy-2,2-bis(p-chlorophenyl)ethane; N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide melting point. 0006-291x/90 Copyright All rights
EDC, x HCl;
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1228
the
m.p.,
aid
Vol.
BIOCHEMICAL
166, No. 3, 1990
our work all
on the
artificial
work
in the
future
structure dated
of
the
tion
and compared
reveal
the
proteins.
to
essential
of
that
of
computer of
the
features
design the
tight
and probably
in general.
antibody
modelling,
designed
of
anti-DDT
polypeptide
protein
site
RESEARCH COMMUNICATIONS
could
and specific
antibody
would
be eluci-
and x-ray
polypeptide. also
The diffrac-
This
may
DDT binding
by
be of great
value.
The purpose anti-DDT (bovine
field
analysis,
A monoclonal
analytical
DDT-binding
DDT binding
by sequence
AND BIOPHYSICAL
of
the
antibodies. serum albumin)
polyclonal respectively.
study
required
the
preparation
Here we describe
the
synthesis
conjugate
and monoclonal The isolated
which
anti-DDT
antibodies
antibodies
MATERIALS
elicited
the
of of
a DDT-BSA
formation
in rabbits
were partially
true of
and mice,
characterized.
AND METHODS
Materials. Kelthan (Figure 1) was obtained from Riedel-de Ha& (Seelze, Germany), DDT, DDE and DDA (Figure 1) were from Serva (Heidelberg, Germany). Protein A-Sepharose was received from Pharmacia, Affi-Gel 10 (for coupling of amines) and Affi-Gel 102 (for coupling of carboxylic acids) were from Bio-Rad Laboratories. 125 I-Protein A was kindly provided by Dr. M. Aguet. Synthesis of the DDT derivative (Figure 2, product 4) used for conjugation with BSA. Kelthan (9.5 mmol) and 3-bromopropionitrile (85 mmol) reacted in 96% H2SO4 (23.5 mmol)(8) to give product 1 (Figure 2) which after precipitation with ice water and recrystallizationofrom ethanol was obtained as white needles in 90% yield, m.p. 171 C. Product 1 (4.0 mmol) was converted to product 2 (Figure 2) by reaction with sodium azide (8.0 mmol) and tetrabutylammonium bromide (0.28 mmol)(9,10) in 3 ml of benzene and dimethylformamide each. Product 2 was isolated from ethanolic solution by precipitation with ether in 62% yield, m.p. 155OC. It was reacted with an equimolar amount of triethyl phosphite in benzene (10,ll) and then with HCl gas to give product 3 (Figure 2) which was precipitated by adding ether. The yield of the white powder obtained was 95%, m.p. 103OC. The last step of the derivatization consisted in the reaction of product 3 (1 mmol) with succinic anhydride (5 mmol) in dry pyridine. After evaporation of the pyridine, extraction of excess succinic anhydride with 0.1 M HCl/ methanol (lO:l), and recrystallization from acetone the target DDT derivative (product 4, Figure 2) was obtained in 53% yield, m.p. 153Oc. The course of the synthesis was followed by elementary analysis (Table l), mass spectrometry, infrared and NMR spectroscopy of the products. In the target DDT derivative (product 4, Figure 2), for example, the protons of the two amides could be distinguished of the amide at the a-carbon gave a singuby 'H-NMR: The proton lett at 8.53 ppm whereas the proton of the amide bond in which the 1229
Vol.
166, No. 3, 1990 Table
1:
Product Product Product Product
1 2 3 4
Numbers
in
P-NH2
by the
40.74 43.96 43.10 46.40
BIOCHEMICAL Elementary
(40.47) (43.78) (42.80) (46.66)
parentheses
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
analyses in Figure
2.83 2.61 3.25 3.51 are
(2.60) (2.79) (3.36) (3.52)
the
of 2
the
2.71 11.90 5.76 5.00
theoretical
products
shown
(2.78) (12.00) (5.87) (5.18)
-44.70 33.05
(44.60) (32.82)
values.
group participates, produced a triplett protons at the adjacent B-carbon.
at
7.83
ppm caused
Preparation of a DDT-BSA conjugate. The DDT derivative (Figure 2, product 4; 18 umol) was activated with excess EDC (N-ethylN'-(3-dimethylaminopropyl)carbodiimide x HCl; 36 umol) (12) in dimethylformamide (1 ml) and added to a solution of BSA (0.3 umol) in 20% aqueous dimethylformamide (5 ml). The turbid mixture was dialyzed against water, lyophilized, resuspended in 0.05 M NH4HC03, and treated with excess cold acetone to precipitate the DDT-BSA conjugate. The yield of the conjugate was 0.16 umol or 54% based on a DDT/BSA molar ratio of 26 as determined by quantitative p-alanine analysis after acid hydrolysis in 6 M HCl at llO°C for 20 h. The DDT/BSA molar ratio could be largely manipulated by the experimental conditions of the coupling reaction (Table 2). Preparation of a DDT-lysozyme conjugate. Hen egg-white lysowas acetylated using acetic anhydride in a 60-fold molar The yield of this step was excess per primary amino group (13). 62%. The reaction between the acetylated lysozyme (6.8 umol) and the p-alanine amide derivative of DDT (B-alanine amide-DDT, Figwas mediated by EDC (four 2.2-mm01 ure 2 (product 3); 21 umol) portions) in 25% aqueous dimethylformamide (22 ml) at pH 5. After 2 h the mixture was dialyzed against 0.1% trifluoroacetic acid and then lyophilized. The conjugate (yield, 56%) contained equizyme
molar
tative
amounts
of
S-alanine
DDT
derivative
and
lysozyme
as
shown
by
quanti-
analysis.
Antibodies against the DDT-BSA conjugate. To raise pOlyClOna1 antibodies, a rabbit was injected with 100 ug of conjugate II
Table
2:
Variability ratio
of
of the DDT derivative-BSA the conjugates
DDT derivative / BSA / EDC molar ratio employed in the coupling reaction 90 60 90
DDT derivative ratio of the conjugate
: 1 : 90 :l : 120 : 1 : 180
10 : 1 26 : 1 41 : 1 1230
molar
/ BSA molar resulting ( 1) ( 11) (III)
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(Table 2) in complete Freund's adjuvant (Difco) and PBS (phosphate-buffered saline), pH 7.2 (0.5 ml each), followed by four monthly booster injections of conjugate II (50 ug each) in incomplete Freund's adjuvant and PBS (0.25 ml each). Blood samples from the ear vein of the rabbit were kept at4OC overnight and then centrifuged. The supernatant sera were stored frozen. Monoclonal antibodies were obtained by the following procedure: Female BALB/c mice received intraperitoneal injections of 40 ug of conjugate II in complete Freund's adjuvant and PBS (0.25 ml each), followed by three monthly booster doses (40 ug each) in incomplete Freund's adjuvant and PBS (0.25 ml each). After the presence of anti-DDT antibodies had been demonstrated in a competitive solid phase RIA (radioimmunoassay), one mouse received an additional injection of 4@ ug of conjugate II in PBS and was sacrificed four days later. The washed spleen cells of this mouse were fused with X63Ag8.653 myeloma cells (14,15) and grown on feeder layers of peritoneal cells in 10% FCS (fetal calf serum; Inotech) in Iscove's modified Dulbecco's medium that gave a positive competi(Gibco) (16). Cells from hybridomas tive solid phase RIA were subcloned twice after limited dilution. Positive clones secreting IgG and IgM anti-DDT antibodies were injected into female BALB/c mice primed with 2,6,10,14-tetramethylpentadecane (Sigma). The ascitic fluids obtained from these animals were dilgted with 2 volumes of PBS and dialyzed against 18% Na2S04 at 22 C overnight. The precipitates formed were centrifuged. A monoclonal IgM anti-DDT antibody was purified as follows: The precipitate was redissolved in PBS and the solution applied to a goat anti-mouse IgM antibody agarose column (Sigma; capacity, 0.4 mg/ml gel). Bound IgM was eluted by 0.1 M glycine0.15 M NaCl, pH 2.4, and had a gel electrophoretic purity of -90%. The solution was neutralized with 1 volume of ten-fold concentrated PBS, dialyzed against PBS, and applied to a DDT-lysozyme column (prepared from EDC-activated DDT-lysozyme conjugate and Affi-Gel 102 using 100 mm01 EDC, 11 umol conjugate, and 50 ml Affi-Gel 102 in 200 ml PBS, QH 5; coupling yield, 38 nmol conjugate per ml gel). The IgM was eluted again by the glycine buffer, DH 2.4, and the purity of the monoclonal antibody was demonstrated by gel electrophoresis (Fiqure 3). Competitive solid phase RIA of DDT and DDT derivatives. Each incubation step on the microtitre plates was followed by 3 washes with PBS. Plates were coated with the DDT-lysozyme conjugate (3.5 ug in 50 ul of PBS/well) at 4OC overnight. Additional protein binding sites were saturated with an emulsion of 1% powdered milk in PBS at 4OC for 6 h. Then 50 ul of a 1:l mixture of diluted antiserum and hapten solution were added per well. After 8 h at room temperature the wells were washed and the residues incubated with 1251-protein A in 1% powdered milk in PBS (50 ul per well containing -100'000 cpm) at 4OC overnight. Finally, the radioactivity bound by the washed wells was counted. Diluted antiserum and hapten solutions were prepared as follows: Antiserum (ascitic fluid) was diluted lOOO-fold with 20% FCS in PBS after FCS had been passed through a commercial protein ASepharose column to remove any IqG. Stock solutions (1 mM) of DDT, p-alanine amide-DDT (Figure 2, product 3), DDE, DDA, and p-chlorotoluene in ethanol were also diluted with 20% FCS in PBS to concentrations between 0.02 and 64 uM. The resulting mixtures were sonicated and stored at room temperature at least 12 h before use. 1231
Vol. 166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION The efficiency could
of our design
be assessed
perties
of
the
occurring
best
designed
bodies
protein.
proteins
would
with
great
value
also
studies
with
DDA).
against
DDA-protein
authors
anti-DDT
Synthesis
of
by the
ting
alkylbromide
3) by the
(Figure
azide
present using
analyses (e.g.
have the
work,
and
DDE and
been prepared first
a semisynthetic
ratio
true DDT-BSA
product
a DDT-lysozyme 1) that
the
analysis
reducing
agent
4 and BSA, conjugate
26)
(molar the
solid
i) that
resul(product
and hydra-
showed that
amine was obtained Staudinger
the
amine
phthalimide
were prepared;
was used in
of
corresponding
2) by the
to carrier
3-bromopropionit-
conversion
structural
as the
coupling
with
of potassium
The desired (product
conjugates of
for
Direct
1) to
action
phosphite
DDT-protein
lysozyme,
conjugates
natural Such anti-
environmental
suitable
iH-NMR and x-ray
triethyl
and ii)
accessible
DDT metabolites
the
(8).
(product
was formed.
(molar
a naturally
antibodies.
1) was combined
reaction
consecutive
intermediate ent
In
DDT derivatives
Ritter
failed;
using
major
were obtained
Kelthan
rile
p-lactam
of
pro-
as immunogen.
proteins.
zine
(17-19).
antibodies
conjugate
those
for
polypeptide
and DDT-binding
The most easily
cross-reactivity by several
structure
seemed to be anti-DDT
be of
Antibodies
a DDT-binding
polypeptide
DDT-binding
DDT-binding
of
by comparing
from
reaction (10).
the (11)
Two differ-
a DDT-BSA conjugate was used as immunogen,
ratio
of product
phase RIAs
3 and
and as affini-
DDE
DDT
HO’
C\
‘0
DDA
DDT-OH (KELTHAN)
formulae of the insecticide Figure 1: Structural metabolites DDE and DDA, and Kelthan. 1232
a
DDT,
its
major
Vol.
166, No. 3, 1990
BIOCHEMICAL
R-OHa +
AND BIOPHYSICAL
[H2S041
NC-CH2-CH2-Br
RESEARCH COMMUNICATIONS
R-NH-CO-CH2-CH2-Br Product -1
)
NaN3 1 R-NH-CO-CH2-CH2-N3 Product -2
13 HCl
succinic anhydride ) pyridine
R-NH-CO-CH2-CH2-NH3+ClProduct -3
R-NH-CO-CH2-CH2-NH-CO-CH2-CH2-COOProduct 4 Figure 2: Synthesis scheme of the DDT derivatives used for conjugation with albumin and lysozyme. In product 4, the p-alanine amide moiety is underlined. aR-OH: DDT-OH (Kelthan, Figure 1).
ty matrix.
The distinct
interference,
in
antibodies
solid
formed
Preparation tion
of
pared
against
for
amide-DDT
10 by displacing agarose.
the
agarose
not
bind
conjugate matrix
length
bound partially
purified
M
acid
or
were not
monoclonal
ted
this
from
as eluant
DDT columns
using
First,
of
resulting
the
spacer
was %20 A,
the
activabetween
column
This
antibodies
by 3 M guanidine
x HCl in
5 mM sodium
0.1 M glycine
and were sufficiently
pure
did
affinity
anti-DDT
in
p-
DDT-lysozyme
102.
IgG and IgM anti-DDT
column
were pre-
to Affi-Gel
EDC-activated
glycol
of
purifica-
properties.
polyclonal
eluted
for
portion
the
out
DDT-BSA complex.
matrices
to Affi-Gel
50% ethylene
However,
of
Then,
covalently
they
the
3) was coupled
DDT moiety
antibodies.
that
affinity
ruled
chromatography,
of
N-hydroxysuccinimide the
conjugates
antibody-binding
was linked
acetic
BSA moiety
2, product
and the
anti-DDT
strongly
2.4,
their
Although matrix
these
Two different
(Figure the
of
and affinity
of
antibodies.
and tested
ted
the
and properties
anti-DDT
alanine
utilization phase RIAs
0.25
hydroxide.
antibodies
could
x HCl/O.l5
M NaCl,
(Figure
so
3) for
be elupH
sequence
analysis. Competitive of
solid
the monoclonal
coated tions
plates of
phase radioimmunoassays
IgM anti-DDT-BSA
was followed
DDT, p-alanine
in
the
amide-DDT
antibody presence (Figure
1233
(Figure
4).
Binding
to DDT-lysozymeof
rising
2, product
concentra3),
DDE, DDA,
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
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97006
-
66200
-
45666
-
29666
-
14366
RESEARCH COMMUNICATIONS
Figure 3: SDS-Polyacrylamide gel electrophoresis of the purified mouse monoclonal IgM anti-DDT antibody. Lane 1, commercial bovine monoclonal IgM (Sigma), reduced with B-mercaptoethanol; lane 2, molecular weight marker proteins; lane 3, purified mouse monoclonal IgM anti-DDT antibody, reduced with B-mercaptoethanol (band near top shows IgM heavy chain, band near bottom IgM light chain). The polyacrylamide gel was 10% cross-linked.
and p-chlorotoluene iS
the
part
alS0
of
binding
of
completely.
the the
Free
DDT and free binding
as
of
a control.
Only
conjugates
with
antibodies
amide-DDT
BSA and lysosyme
to
the
DDT had 70% of
the
activity
of
There
was very
by the
which
repressed
DDT-lvsozyme-coated
DDE was 40% as active. DDA or p-chlorotoluene
P-alanine
elates
P-alanine
amide-
little
monoclonal
if
any
anti-DDT-BSA
antibody. Taking
into
account
the
solubility
of
amide-DDT>DDA>DDT=DDE),
the
the monoclonal
was largely
group
of
the
antibody DDT molecule;
The fact
cross-reactivity part the
of
the
ure
with epitope
derivative.
was applied of
that
To
p-alanine
and the
showed that
p-alanine
the of
DDT-lysozyme p-alanine
alone. DDT with
It
amide
on the
p-alanine dilute
p-alanine
RIA carried amide did
conjugate and
was concluded the
antibody
antibody
anti-DDT-BSA
as described.
not
inhibit
and that
DDT was
that
the
indistinguishable
the
lack
antibody 1234
of
by ethylenic
showed 100% could
mean that
amide portion ascitic
mixt-
micro-
The results binding
of from
to
the mixture that
100% cross-reactivity
was most likely
of
fluid
a 1:l
antibody
behaviour
C13C-
binding
amide and ii)
out
that
the
group
amide-DDT
possibility,
i)
suggested
against
of this
anti-DDT-BSA
this
RIAs
directed
p-alanine
was located with
the
(B-alanine
amide and DDT to a DDT-lysozyme-coated
plate
titre
the
together
haptens
(DDA) abolished only
test
of
replacement
C12C= (DDE) and carboxylate stepwise.
results
the
caused
of
DDT
of by the
Vol.
BIOCHEMICAL
166, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 4: Solid phase radioimmunoassay of the cross-reaction of DDT and DDT derivatives with a monoclonal IgM anti-DDT-BSA antibody on a DDT-lysozyme-coated microtitre plate. Details of the assay procedure are given in MATERIALS AND METHODS. Using an IgM antibody required incubation of the DDT-lysozyme - IgM - coated plate with the IgG fraction of rabbit anti-mouse Ig antiserum (Nordic Immunology) prior to the addition of 1251-protein A. A 1:l mixture of diluted ascitic fluid and 20% FCS in PBS was used as positive control (100% radioactivity), 20% FCS in PBS alone was used as negative control (0% radioactivity). DDT-lysozyme binding of the anti-DDT-BSA antibody was studied in the presence of DDA or p-chlorotoluene m, DDE (a), DDT (O), and p-alanine amide-DDT (0).
limited
solubility
concentrations
above
Competition bodies
or aggregation
solid
similar
0.0035
favourably
with
chromatography
mg/l the
via
its
carboxyl
DDT-BSA antibodies well
DDT in aqueous
polyclonal
limit
nM or 0.035 group
of
mg/l).
method
high
performance
Antibodies
the
specificity
anti-
as low as
by this
DDA was directly lacked
at
anti-DDT-BSA
DDT concentrations
were detectable
in which
buffers
4).
results.
detection
(-100
a DDA-BSA conjugate tein
~1 uM (Figure
phase RIAs using
gave very
%13 nM or
of
comparing liquid
raised
linked
against
to the of our
and bound DDT, DDE, and DDA almost
pro-
anti-
equally
(19). ACKNOWLEDGMENTS
We thank Dr. P. Wipf for advice during the synthesis of the DDT derivatives, Dr. R. Prewo for the x-ray structural analysis of the p-lactam derivative of DDT, Dr. M. Aguet and Martina Metzler for help during the preparation of the monoclonal antibodies, and the Schweizerische Nationalfonds and the Roche Research Foundation for financial support of this work. 1235
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4.
Gutte, B., Daumigen, M., and Wittschieber, E. (1979) Nature 281, 650-655. Moser, R., Thomas, R-M., and Gutte, B. (1983) FEBS Lett. 157, 247-251. Mutter, M. (1988) Trends Biochem. Sci. 13, 260-265. Lear, J.D., Wasserman, Z.R., and DeGrado, W.F. (1988) Science 240,
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